The sarcoplasmic reticulum Ca 2؉ -ATPase transports Ca 2؉ using the chemical energy derived from ATP hydrolysis. Part of the chemical energy is used to translocate Ca 2؉ through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca 2؉ gradient across the vesicle membrane. In the absence of gradient (leaky vesicles) the amount of heat produced/mol of ATP cleaved is half of that measured in the presence of the gradient. After formation of the gradient, part of the ATPase activity is not coupled to Ca 2؉ transport. We now show that NaF can impair the uncoupled ATPase activity with discrete effect on the ATPase activity coupled to Ca 2؉ transport. For the control vesicles not treated with NaF, after formation of the gradient only 20% of the ATP cleaved is coupled to Ca 2؉ transport, and the caloric yield of the total ATPase activity (coupled plus uncoupled) is 22.8 kcal released/mol of ATP cleaved. In contrast, the vesicles treated with NaF consume only the ATP needed to maintain the gradient, and the caloric yield of ATP hydrolysis is 3.1 kcal/mol of ATP. The slow ATPase activity measured in vesicles treated with NaF has the same Ca 2؉ dependence as the control vesicles. This demonstrates unambiguously that the uncoupled activity is an actual pathway of the Ca 2؉ -ATPase rather than a contaminating phosphatase. We conclude that when ATP hydrolysis occurs without coupled biological work most of the chemical energy is dissipated as heat. Thus, uncoupled ATPase activity appears to be the mechanistic feature underlying the ability of the Ca 2؉ -ATPase to modulated heat production.The main physiological role of the sarcoplasmic reticulum Ca 2ϩ -ATPase is to remove Ca 2ϩ from the cytosol thus controlling the transition between muscle contraction and relaxation (1-4). The ATPase uses the energy derived from ATP hydrolysis to transport Ca 2ϩ across the membrane thus converting chemical energy into osmotic energy. The catalytic cycle of the Ca 2ϩ -ATPase can be reversed, during which Ca 2ϩ leaves the vesicles through the ATPase, and coupled to the Ca 2ϩ efflux, ATP is synthesized from ADP and P i using the energy derived from the Ca 2ϩ gradient (3, 4 -9). Contrasting to the removal of Ca 2ϩ from the medium, there is no evidence indicating that the reversal of the Ca 2ϩ pump can play a role in muscle physiology. In recent years, two more properties of the Ca 2ϩ -ATPase were discovered that apparently are of no physiological relevance, the uncoupled Ca 2ϩ efflux (10 -12) and the uncoupled ATPase activity (13-15). During transport, part of the Ca 2ϩ accumulated by the vesicles leaks to the medium through the ATPase, but this efflux is not coupled to ATP synthesis. The uncoupled Ca 2ϩ -ATPase activity was described by Yu and Inesi (13), who observed that the progressive rise in the Ca 2ϩ concentration in the vesicle lumen promotes the hydrolysis of ATP without concomitant Ca 2ϩ translocation through the membrane. Depending on the conditions used, the...